1. Field of the Invention
The invention relates to chill preservation and cryopreservation compositions which control the induction of apoptosis in preserved cells. Particularly, the invention relates to methods of preserving cells, tissues and organs at hypothermic temperatures utilizing intracellular-type hypothermic maintenance and preservation media compositions containing reagents which control the induction of gene regulated cell death (apoptosis). The invention further relates to methods of cryopreserving cells with an intracellular-type hypothermic maintenance and preservation composition containing reagents which control the induction of apoptosis in preserved cells.
2. Description of Related Art
Non-blood based solutions useful for preserving organs, tissues and cells are well-known in the art. For example, blood substitutes for preserving tissues and organs for transplantation are disclosed by U.S. Pat. No. 4,920,044 to Breton and U.S. Pat. Nos. 4,879,283 and 4,798,824 to Belzer. U.S. Pat. No. 4,923,422 to Segall et al discloses a four solution blood substitute useful in hypothermic preservation of isolated organs for use in transplantation procedures. Typically, cold storage of human tissues at 4.degree. C. in a conventional preservation solution results in cellular debris collecting in the culture medium surrounding the tissue samples within the first several days of storage. Many cells shrink after a few days at 4.degree. C. and display ultrastructural damage. These cells and their tissues are not viable after cold-storage as a result.
Hypothermic storage solutions have been developed for shipping and storing organs, tissues and cells. Cells are immersed in these solutions and kept at or near 4.degree. C., where they remain in a state of prolonged hypothermia. The growing number of engineered tissues has increased scientific interest in developing solutions for cold-store tissues at 4.degree. C. on a short-term basis, but without the use of cryopreservation. Cold storage or hypothermic solutions, such as VIASPAN.RTM. [also called University of Wisconsin (UW) solution], EURO-COLLINS and others that have been used for organ storage (1), are now being tested for their abilities to keep cells and tissues in a state of hypothermia.
The importance of optimizing a cold-storage solution for cell/tissue storage cannot be overstated. Pahernik et al. (2) has noted the importance of large-scale stocking facilities for hepatocytes to develop bioartificial livers. This group points to chill preservation solutions as a milder alternative to cryopreservation since cryopreservation often results in a major loss of cell function. Fisher et al. (3) have tested VIASPAN.RTM., EURO-COLLINS, Sacks+prostacyclin and V-7 for their abilities to cold-preserve tissue slices. The use of these chill preservation solutions for tissue slice preservation at temperatures between 5.degree. C. and 0.degree. C. would effectively increase the shipping/storage life of tissue sections, thereby decreasing the number of animals used in cosmetic and pharmaceutical testing. Another hypothermic solution developed by Taylor et al. (4) has been shown to protect engineered human epidermis at 4.degree. C. for over a week (5). This bioartificial skin produced by MatTek Corporation (Ashland, Mass.) continues to differentiate at 37.degree. C. after a week in an intracellular-like hypothermic blood substitute solution, HYPOTHERMOSOL.RTM., at 4.degree. C. Chill preservation of single cells has also been investigated by several groups For example, normal human epidermal keratinocytes can be maintained for periods exceeding one week at 4.degree. C. in HYPOTHERMOSOL.RTM. versus only 24 hours in normal growth media (10). Similar results were obtained following the hypothermic storage of EPIDERM.RTM., an engineered human epidermis used in product safety testing (5).
Hypothermic preservation solutions such as UW solution have been used for the preservation of heart (11-13), liver (1-16), lung (17-19), kidney (20), and small intestine (21). These procedures have been recently extended to the area of in vitro toxicology. Fisher et al. (3) have shown the importance of storing living liver and kidney slices used as animal alternatives in hypothermic preservation solutions. Cook et al. (5) have shown that HYPOTHERMOSOL.RTM. can be used for the cold-storage of engineered human epidermis at temperatures between 5.degree. C. and 0.degree. C. This observation is important because HYPOTHERMOSOL.RTM. could extend the shelf life of bioartificial human epidermis used as an alternative to animal testing. Alternatively, HYPOTHERMOSOL.RTM. or an alternative solution could be used to store human epidermis prior to grafting in clinical cases. While bioartificial livers are not currently in use, Pahernik et al, (2) has pointed, nonetheless, to the importance of banking liver cells for liver tissue engineering. However, the effectiveness of current chill preservation solutions is limited by cellular processes including gene regulated events which promote cell death during cold storage.
While cold storage of cells and tissues at about 5.degree. C. to 0.degree. C. is suitable for short periods of time, long-term storage of cells requires cryopreservation at temperatures between the onset of freezing and the temperature of liquid nitrogen (-196.degree. C.). Cryopreservation, the maintenance of biologics including numerous cell lines at or near liquid nitrogen temperature, is a critical methodology necessary to support biomedical research. Preservation protocol development has focused on overcoming freeze-induced cell death due primarily to intracellular ice formation and chemo-osmotic stress, which result in plasma membrane disruption and subsequent necrosis (43). Accordingly, numerous investigators have cryopreserved cellular systems in cryoprotective agents, such as dimethyl sulfoxide (DMSO) or glycerol, contained in an extracellular-like carrier solution such as standard cell culture media. This strategy was first described following the partial preservation of bovine spermatozoa (44). Subsequently, numerous investigators have cryopreserved red blood cells (45), white blood cells (41), gametes and embryos (34), and simple tissues including heart valves (32) with limited success.
Traditional approaches to cryopreservation rely on the addition of molar concentrations of penetrating cryoprotectants contained in either isotonic or hypotonic media. This approach rarely considers the ionic balance, buffering capacity or other factors thought to be necessary to avoid the consequences of hypothermic stress. This situation may be problematic in view of the fact that cells during slow cooling (.about.1.degree. C. min.sup.-1) remain in a hypothermic state until the intracellular contents vitrify during liquid nitrogen (LN.sub.2) quenching step of the preservation protocol. Post-thaw viability of cells is low using traditional methodology, such as freezing cells in solutions of culture media diluted with 5% to 10% DMSO. A post thaw survival rate of only about 30% of a frozen cell population is common when traditional protocols are followed. The high loss of cell viability after thawing is thought to be due to crystal formation and cellular dehydration that occurs during freezing.
The majority of investigators have assessed viability within a few hours of thawing using dye exclusion assays but provide little information on long term survival (3,27). For example, human hepatocytes assessed using a trypan blue dye exclusion assay yield approximately 67% survival following 24 hour storage but only 49% after 14 days storage at -80.degree. C. (29). More recently, Adams et al. (26) have shown that hepatocyte preservation outcome can be improved by utilizing VIASPAN.RTM. (an intracellular-like, hypothermic storage solution) as the carrier solution fortified with fetal bovine serum and DMSO.
Nagle et al. (40) presented evidence suggesting that a molecular-based mode of cell death was associated with hypothermic storage and that cell death could not be accounted for by solely addressing the known mechanisms of chill-induced cellular injury. Parks (42) noted that the extent of hypothermic exposure experienced by cells during the preservation process appears to be a major factor affecting the success of cryopreservation. Recently, it has been reported that apoptotic cell death occurs in cardiomyocytes as a result of hypothermic exposure (39). Apoptosis, or programmed cell death, is a gene-activated event that occurs as a normal consequence of development (37), as well as a result of cellular stress (30). Hollister et al. (36) demonstrated that apoptosis is a significant component of cell death in a human prostate cancer cell line (PC3) exposed to subfreezing temperatures as low as -75.degree. C. These data point to apoptosis as a possible contributing factor in cryopreservation failure.
Cell death may occur through apoptosis or necrosis (for a review see 24). Necrosis or pathological cell death is characterized by the loss of cell membrane integrity resulting in cell swelling and is caused by a number of pathological agents. DNA in cells that undergo necrosis is cleaved in a random fashion. Thus, the DNA from cells that have undergone necrosis appears as a continuous smear when subjected to gel electrophoresis. Apoptosis, is gene activated and is characterized by shrinking cells, intact plasma membranes, and the formation of apoptotic bodies. DNA in cells undergoing apoptosis is cleaved in a non-random fashion, forming a ladder-like pattern upon gel electrophoresis. The principal mode of cell death for many cells stored under hypothermic conditions for periods longer than 1-2 days in conventional preservation solutions appears to be necrosis, not apoptosis. The relationship between short term storage cold-storage and apoptosis is unclear.
The precise cellular mechanisms regulating apoptosis are not completely known. However, many portions of the apoptotic pathway have been delineated to date. Alteration of the ionic environment may be necessary to activate or inhibit the endonucleases relevant to the process of apoptotic nuclear degradation. For example, physiologic concentrations of Zn.sup.++ are known to inhibit DNA fragmentation and apoptosis. Treatment of certain cells with inhibitors of macromolecular synthesis, such as actinomycin to block RNA synthesis or cyclohexamide to block protein synthesis, induces apoptosis. Completion of the apoptotic process appears to depend upon the regulated expression of various gene products associated with the promotion or suppression of gene activated cell death, particularly gene products involved with cell cycle regulation. For example, overexpression of the cell-death inhibiting agents Bcl-2 and Bcl-xL prevents the release of cytochrome C. Cytochrome C is thought to activate the caspases, a group of proteases known for cleaving substrates responsible for the changes associated with apoptosis. Enhanced levels of Bax, a pro-apoptotic member of the Bcl-2 family, promotes cytochrome C release and subsequent apoptosis of cells. Specific regulation of the early response genes c-myc, c-jun and c-fos may promote either cell growth or cell death, depending upon the circumstances surrounding their expression. Thus, programmed cell death involves an intricate cascade of cellular events.
The cold-storage efficacy of numerous cell preservation solutions has been examined using a plethora of assays. Such assays include enzyme synthesis (2), potassium content (3), trypan blue exclusion (7), neuronal outgrowth and myelination (8), contraction (11), ATP content (13), and ultrastructure (17). As pointed out previously (10), these assays can be considered either viability or functional assays. Thus, the maltose tolerance test used by Katz et al. (21) on the small intestine could be considered a functional assay; whereas the trypan blue test used by Rodriguez et al. (7) is strictly a viability assay. The ALAMAR BLUE.TM. assay used in the course of the study reported herein is a viability assay and does not indicate whether or not test cells are functioning in a tissue specific manner. Yet one of the key attributes of the ALAMAR BLUE.TM. assay that is not shared by most of the other viability or function assays described previously is the ability of ALAMAR BLUE.TM. to be used repetitively, day after day, as a non-toxic indicator. This has been shown to be critically important to some cold-stored tissues such as human skin cells.
Hypothermic preservation of cells, tissues and organs is important for the storage of cell lines useful for biomedical research, preservation of forensic samples, storage of medical samples (such as biopsy material and samples for in vitro fertilization, etc.), and for maintenance of cells, tissues and organs for transplantation. For example, the preservation of pancreatic islet cells is critically important to the future of clinical cell transplantation. Korbutt and Pipeleers (6) have shown that rat pancreatic beta cells can survive a 96 h storage period at 4 .degree. C. in VIASPAN.RTM.. Other groups (7) have shown that hepatocyte suspensions can be stored in UW solution. Levi et al. (8) have tested UW solution as a potential cold-storage solution for nerve cells in the consideration of a future nerve bank as a source of material to repair damaged nerves. Gall bladder biliary epithelial cells have also been tested in cold-storage solutions (9). Finally, it has shown that isolated human skin cells growing in culture can be very well preserved in HYPOTHERMOSOL.RTM. (CMS, Rockville, Md.)(10).
Many of these studies tested a variety of additives that enhanced the cold-storage efficacy of hypothermic preservation solutions, but none have done so in view of how cells die following long-term storage in such preservation solutions. A variety of supplements have been added to hypothermic-storage solutions to test their potential as beneficial adjuncts. Rodriguez et al. (7) have shown that the addition of glutathione to UW solution is beneficial to the preservation of hepatocytes. Lopukhin et al. (11) have shown that the addition of 2,3 butanedione monoxime to UW solution increases the storage life of preserved heart. Glycine, however, was found to not be beneficial when added to UW solution (14). Of particular interest, however, is the addition of antioxidants. Lazaroids, for instance, have been shown to increase the cold storage efficacy of UW solution (15,21). Glutathione has been shown to enhance the storage of lung when added to VIASPAN.RTM. (19).
Prior studies employing both chill preservation and cryopreservation techniques indicate that a barrier exists for each cell population which precludes full recovery from cold storage. The emergence of engineered tissues utilized in clinical applications, basic research and product safety testing has heightened the demand for improved chill preservation and cryopreservation techniques to ensure the viability of both cells and engineered multi-layered tissues subjected to hyopthermic storage conditions (38).